View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Journal of Genetic Engineering and Biotechnology (2016) 14, 247–251 HOSTED BY Academy of Scientific Research & Technology and National Research Center, Egypt Journal of Genetic Engineering and Biotechnology www.elsevier.com/locate/jgeb ORIGINAL ARTICLE Common variants in TCF7L2 and CDKAL1 genes and risk of type 2 diabetes mellitus in Egyptians Dalia El-Lebedy *, Ingy Ashmawy Department of Clinical and Chemical Pathology, Medical Research Division, National Research Centre, Cairo, Egypt Received 17 July 2016; revised 21 September 2016; accepted 17 October 2016 Available online 5 November 2016 KEYWORDS Abstract In this work we studied association of common variants in transcription factor 7-like 2 Type 2 diabetes mellitus; (TCF7L2) and cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) TCF7L2; genes with type 2 diabetes mellitus (T2DM) in Egyptians. CDKAL1; Subjects and methods: This is a case–control study; 180 T2DM patients and 210 control subjects Polymorphism were genotyped for TCF7L2 rs7903146 and rs12255372 and CDKAL1 rs7756992 single nucleotide polymorphisms (SNPs) by TaqMan method on real time polymerase chain reaction system (real time-PCR). Results: TCF7L2 rs12255372 and rs7903146 associated with T2DM (p = 0.0001 and 0.003; respectively). The rs12255372 variant T allele associated with 2-fold increased risk for T2DM and TT genotype carriers were at 3.58-folds higher risk to develop T2DM than wild genotype (GG) carriers. Meanwhile, rs7903146 variant T allele associated with 1.6-fold increased risk for T2DM and TT genotype carriers were at 2.3-folds higher risk than wild genotype (CC) carriers. Both TCF7L2 SNPs significantly associated with T2DM under additive and dominant models and after adjustment for other covariates. On the other hand, CDKAL1 rs7756992 showed no sig- nificant association with T2DM under any genetic model. Both TCF7L2 SNPs were in strong LD (P = 0.02; D0 = 0.85). Taking common TCF7L2 rs12255372/rs7903146 GC haplotype as reference, multivariate analysis confirmed the association of rs12255372 T allele-containing haplotypes (TC and TT) with T2DM. Haplotype TC associated with 6.32 times-higher risk for T2DM (95% CI = 0.55–76.17, Pc = 0.04) followed by haplotype TT which associated with 3.88 times-higher risk for the disease (95%CI = 1.09–13.76, Pc = 0.03). Conclusion: TCF7L2 rs12255372 and rs7903146 common variants associate with T2DM risk in Egyptians. Ó 2016 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). 1. Introduction * Corresponding author at: Department of Clinical and Chemical Pathology, Medical Research Division, National Research Centre, Al- Bohouth Street, Cairo 12311, Egypt. Type 2 diabetes mellitus (T2DM) is a metabolic disorder E-mail address: [email protected] (D. El-Lebedy). caused by decreased insulin sensitivity and impaired insulin Peer review under responsibility of National Research Center, Egypt. secretion due to pancreatic beta cell defect [1]. In addition to http://dx.doi.org/10.1016/j.jgeb.2016.10.004 1687-157X Ó 2016 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 248 D. El-Lebedy, I. Ashmawy environmental and lifestyle risk factors, genetic factors play an Control subjects had normal glucose tolerance confirmed important role in disease pathogenesis [2]. So, identification of by fasting plasma glucose (FPG) < 126 mg/dl or 2 h plasma genetic architecture of T2DM is of great interest for risk pre- glucose (PPG) < 200 mg/dl or random plasma glucose (ran- diction and preventive interventions. dom blood sugar) (RBG) < 200 mg/dl, and no first-degree T2DM represents a global major healthcare burden [3]. family history of diabetes. Informed consent was obtained According to the International Diabetes Federation (IDF), from all subjects and the study protocol was approved by Egypt is in the world 8th place in terms of diabetes incidence, the Ethics Committee of the National Research Centre. affecting up to 9.3% of population, and due to a rapidly Clinical examination was applied including measurement of increasing and aging population, Egypt will have the highest systolic blood pressure (SBP) and diastolic blood pressure number of people with diabetes in the region by 2025 [4]. (DBP). Anthropometric measurements (weight and height) According to genome-wide association studies (GWAS) were collected and used for BMI calculation according to the and meta-analysis; more than 50 gene variants were identified standard formula BMI = weight (kg)/[height (m)]2. to be associated with T2DM. Two common variants in tran- scription factor 7-like 2 (TCF7L2) gene on chromosome 2.2. Biochemical analysis 10q25.3 have brought the most attention and were reported as the strongest genetic risk factor for T2DM [5]; a C-to-T sub- Blood samples for biochemical screening tests were obtained stitution in intron 3 (IVS3C > T, rs7903146) [6–8] and a G-to- from all subjects after a 12 h overnight fast. FPG, TC (total T substitution in intron 4 (IVS4G > T, rs12255372) [6,9]. The cholesterol), TG (triglycerides), HDL-C (high density lipopro- exact mechanism through which TCF7L2 variants affect the tein cholesterol), LDL-C (low density lipoprotein cholesterol) risk for T2DM is still unclear. It has been postulated that and HbA1c (glycated Hb) were assayed on Cobas c311 auto TCF7L2 gene variants may indirectly alter glucagon-like pep- analyzer (Roche Diagnostics, Germany). tide 1 (GLP-1) levels, an insulinotropic hormone which plays a critical role in blood glucose homeostasis [10]. 2.3. Genotyping analysis One of the loci most consistently associated with T2DM risk is the intronic variant within the cyclin-dependent kinase Genomic DNA was extracted from peripheral blood using 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) QIAamp DNA extraction kit (Qiagen Hilden, Germany) gene; A-to-G substitution in intron 5 (IVS5A > G, according to the manufacturer’s protocol. TCF7L2 gene SNP rs7756992) [11]. rs7903146 (assay ID: C_29347861_10) and SNP rs12255372 CDKAL1 gene, located on chromosome 6p22.3, encodes a (assay ID: C_291484_20) and CDKAL1 SNP rs7756992 (assay 65-kD protein (CDKAL1) which is implicated in beta cell dys- ID: C_2504058_20) were genotyped by TaqMan allelic dis- function and T2DM susceptibility [12]. CDKAL1 might regu- crimination method on ABI 7500 real-time PCR system late insulin secretion induced by cyclin-dependent kinase 5 (Applied Biosystems, Foster City, CA). All primers and probes (CDK5) by binding to the CDK5 activator p35 [13–15]. It was were designed by Applied Biosystems (Foster City, CA). For postulated that down regulation of CDKAL1 expression might genotyping quality control, 10% of samples were randomly increase the activity of CDK5 [16]. However, the exact patho- selected and measured in duplicates and the concordance rate genesis of how CDKAL-1 modulates insulin release in pancre- was 100%. atic beta cells and the susceptibility to T2DM by interactions between these proteins still need more investigations [17]. 2.4. Statistical analysis Association studies of these genetic variants with T2DM risk gave inconsistent results in populations from different eth- nic origins, which have been attributed to ethnic variations, Data were analyzed using SPSS version 16.0 for Windows linkage disequilibrium pattern, as well as other non-genetic (Chicago, IL, USA). Data were expressed as mean ± SD for factors [18]. This is the first study to investigate the association continuous variables and as percentages of total for categorical of TCF7L2 rs7903146 and rs12255372 and CDKAL1 variables. Intergroup significance was assessed by Student’s t- 2 rs7756992 variants with T2DM risk in Egyptians. test for continuous variables and v test for categorical vari- ables. The Hardy–Weinberg equilibrium was estimated by v2 2. Subjects and methods test. Chi-square was used to test the difference in alleles and genotypes frequency between groups. The Bonferroni correc- 2.1. Subjects tion method was applied for multiple testing. Associations of genotypes and alleles with T2DM were evaluated by logistic regression after adjustment for other covariates. Odds ratios A total of 390 subjects, including 180 unrelated T2DM (ORs), 95% confidential intervals (CIs) and P values were cal- patients and 210 healthy controls, were enrolled in the current culated. P value less than 0.05 was considered significant. study. T2DM patients were recruited from the outpatient clinic of the National Diabetes & Endocrinology Institute. Diagnosis 3. Results of diabetes based on the diagnostic criteria of the American Diabetes Association 2014 [19], i.e. fasting plasma glucose 3.1. General characteristics of the study subjects (FPG) P 126 mg/dL or 2 h plasma glucose (PPG) P 200 mg/ dl or random plasma glucose (RBG) P 200 mg/dl. Exclusion criteria were type 1 diabetes (T1DM), maturity onset diabetes The study included 390 subjects classified into 180 patients of the young (MODY), or type 2 diabetes diagnosed before the with T2DM and 210 control subjects. Their age ranged from age of 30 years. 40 to 60 (years). Clinical and biochemical data are shown in TCF7L2 and CDKAL1 gene variants and T2DM in Egyptians 249 Table 1. Significant differences between patients and controls results, while some replication studies confirmed this were identified in gender, BMI, SBP, DBP and lipid profile association, other studies failed to find significant association, parameters; accordingly, those were the potential covariates proposing an ethnic variability. that were controlled for in the subsequent analysis. TCF7L2 gene variants have been replicated in many ethnic groups and were associated with T2DM [5], including 3.2.